Health control, diagnosis of disease and/or monitoring of treatment of disease often involves measurement of various parameters. One parameter is the concentration of a certain substance, such as oxygen, within at least part of an organism. Local tissue oxygenation is an important parameter in the diagnosis and treatment of a wide range of diseases. Measurements of the amount of oxygen present in a specific part of a subject are, for instance, carried out during peri-operative monitoring in the operating room and intensive care and for diagnosis of a wide range of clinical disorders in which tissue oxygenation lies central to the development and cure of disease. Examples include diagnosis of cardiovascular disease, monitoring healing of decubitus and diabetic wounds, monitoring hyperbaric correction of radiation wounds and assessment of success of bypass surgery. Monitoring of tissue oxygen pressure (pO2) during critical illness is considered a major need in the adequate treatment of intensive care patients (Siegemund et al., 1999). Assessment of tumor oxygenation is an example wherein measuring of local tissue oxygenation is helpful for the choice of treatment, as oxygen is an important determinant for success of radiotherapy. Hence, the concentration of oxygen in a tumor is preferably determined in order to determine whether radiotherapy is recommended. Local oxygen measurements are also applicable for the assessment of organ viability for transplantation.
Dioxygen is a molecule of utmost biological importance because of its role as the primary biological oxidant. Therefore, oxygen plays a key role in the oxidation/reduction reactions that are coupled to cellular respiration and energy supply. Adequate measurement of oxygen concentrations in biological samples, such as cells, tissues and whole organs, is important to gain insight in the determinants of oxygen supply and utilization under normal and pathological conditions. It is interesting to note that the clinical interest in methods providing information about blood-flow and oxygen delivery at the organ or sub-organ level (e.g., microcirculatory) is growing. This is, amongst other things, because of increasing insight into the role of the microcirculation in pathogenesis, and the importance of adequate tissue perfusion as end-point of treatment (Siegemund et al., 1999).
Various techniques have been developed for direct or indirect oxygen measurements in tissue, each having its specific advantages and disadvantages (for a review on this subject, see J. M. Vanderkooi et al., 1991). Conventionally, measurements of tissue oxygenation have been made by use of oxygen electrodes and spectrophotometry of the hemoglobin or myoglobin molecule. Reflection spectrophotometry records the difference in absorption and scattering between a standard reference sample and a tissue sample. The method is based on the illumination of a tissue sample by light with a known spectral content and detection of the diffusely reflected light from the tissue at several different wavelengths. The spectral difference between illumination light and detected light contains information about the wavelength-dependent absorption and scattering within the tissue. The reference sample, used for correction of non-ideal apparatus behavior, can be anything with well-known absorption properties, but a white sample (no absorption) is mostly used. The relative tissue absorbency [Er(tissue)] can be described by the following equation:[Er(tissue)]=log(Ir(standard)/Ir(tissue))  (1)where Ir(standard) and Ir(tissue) are the intensity of the diffusely reflected light from the white standard and the tissue, respectively. Since the absorption spectra of oxygenated and deoxygenated hemoglobin show marked differences that can easily be detected by RS, this technique is widely used for measurement of hemoglobin saturation in tissue. In order to derive more or less quantitative data with RS, it is necessary to take into account the influence of tissue optical parameters other than the hemoglobin related ones. Different approaches for developing an appropriate analysis algorithm are possible. One described approach is based on the use of isobestic points (the intersection points of the curves of oxygenated and deoxygenated hemoglobin) as reference points within the calculation (Sato, 1979). Dummler used a somewhat different approach for his derivation of an algorithm (Dummler, 1988) based on the two-flux theory of Kubelka and Munk (Kubelka, 1931; Kessler, 1992). The EMPHO, the Erlangen Micro-lightguide spectrophotometer (Frank, 1989) (EMPHO II, Bodenseewerk Gerätetechnik, Überlingen, Germany) and the O2C (Lea Medizin Technik, Giesen, Germany) are spectrophotometers using improved Dummler algorithms for hemoglobin saturation measurements.
The drawback of these conventional techniques is that they are either mechanically disruptive (insertion of oxygen electrodes) or qualitative (spectrophotometry). These constraints have led to the development of alternative methods. One of the most promising techniques in this respect has been the use of oxygen-dependent quenching of phosphorescent dyes for measurements in the microcirculation (Vanderkooi et al., 1987; Sinaasappel & Ince, 1996; Sinaasappel et. al., 1999).
Wilson and Vanderkooi (Vanderkooi, 1987) introduced the oxygen-dependent quenching of phosphorescence of metallo-porphyrin compounds for biological oxygen concentration measurements. The technique is based upon the principle that a metallo-porphyrin molecule that has been excited by light can either release this absorbed energy as light (phosphorescence) or transfer the absorbed energy to oxygen (without light emission). This results in an oxygen-dependent phosphorescence intensity and lifetime. The relationship between the lifetime and the oxygen concentration is given by the Stern-Volmer relationship (Vanderkooi, 1989). Calibration constants associated with the Stern-Volmer relationship allow oxygen concentrations to be calculated from the measured lifetimes. The measurement of lifetimes allows quantitative measurements without the influence of tissue optical properties.
For in vivo measurements, Pd-porphyrin is bound to albumin to form a large molecular complex that, after injection into the circulation, remains confined, at least for a certain time, inside the blood vessels. This allows microvascular pO2 measurements to be made using a phosphorimeter. A phosphorimeter is a device that measures the phosphorescence decay after a pulse of light (time-domain device) or determines the phase shift between a modulated excitation source and the emitted phosphorescence (frequency-domain device). Several of these systems have been described in literature (Mik, 2002; Coremans, 1993; Sinaasappel, 1996; and Vinogradov, 2002).
Attached to a microscope, phosphorescence lifetime measurements allow the measurement of pO2 in single blood vessels in the microcirculation. Use of fiber phosphorimeters allows measurement of microvascular pO2 (μpO2) without having to resort to microscope techniques. A fiber phosphorimeter has been developed for measurement of μpO2 in large animal models of shock and sepsis (Sinaasappel, 1999; Van Iterson, 1998), as well as in mice (Van Bommel, 1998), and the analysis of the decay kinetics has been improved to provide more reliable calculation of pO2 values from the decay kinetics (Mik, 2002). A multi-channel implementation of this phosphorimeter allows simultaneous detection of μpO2 at different sites and different organs. In general, the use of multi-fiber technology is, besides imaging, a way to detect special information in optical spectroscopy. FIG. 1 schematically shows an example of a frequency-domain phosphorimeter of which the light source is a very cost-effective light-emitting diode (LED).
An advantage of lifetime measurements is the independence of the concentration of the chromophore, making quantitative measurements possible in vivo, where the precise concentration of the chromophore cannot be predicted. An important drawback of this technique is, however, that it relies on injection of palladium-porphyrin into the circulation, making this technique unsuitable for clinical settings because of long-term toxicity. The use is limited to pre-clinical applications. Moreover, this technique is only suitable for measuring oxygen levels in the microcirculation. Since the molecules are large and cell-impermeable, this technique cannot be applied for intracellular oxygen measurements without disrupting the intracellular compartment by micro-injection (Hogan, 1999).
A kind of semi-quantitative oxygen measurement using non-specific protein phosphorescence has been used for oxygen measurements in mitochondrial suspensions. This was based on oxygen-dependent quenching of the phosphorescence of the amino acid tryptophan (Vanderkooi et al., 1990). Unfortunately, this phosphorescence cannot be used for quantitative oxygen measurements because of the complex decay kinetics arising from the different tryptophan-containing proteins (Vanderkooi et al., 1987b). The use of tryptophan phosphorescence for in vivo applications is furthermore limited because of the excitation in the UV region (283 nm), resulting in extremely shallow penetration depths in tissue, besides the well-known photo-toxicity of this high energetic light.
Although both oxygen-dependent quenching of phosphorescence and hemoglobin saturation measurements give information about the microvascular oxygenation status, they do not provide a direct measurement of the adequacy of tissue oxygenation. The latter is highly dependent on factors like tissue oxygen consumption and diffusion distances within the tissue. Additional measurements of, e.g., oxygen extraction and CO2 production are, therefore, often required.
More direct spectroscopic determinations of tissue oxygenation are also possible. One of the oldest, and most widely used, is NADH-fluorimetry. The measurement of tissue bioenergetics is commonly used for measurement of the adequacy of tissue oxygenation. Oxidative phosphorylation occurring in the mitochondria of cells is the main site for the production of ATP. In the final step of the electron transport chain, reduced pyridine nucleotides (NADH) is oxidized to NAD+ and H2O, utilizing molecular oxygen. In contrast to NAD+, NADH emits blue fluorescence (around 450 nm) when illuminated with ultraviolet light (around 360 nm). This allows spectroscopic determination of relative tissue NADH levels. The fluorescence intensity of NADH is, therefore, an optical indicator of cellular metabolism.
Measurement of the fluorescence intensity of endogenous mitochondrial NADH in situ can thus be used as a direct measure of tissue bioenergetics. Since for the conversion of mitochondrial NADH to NAD+ the availability of molecular oxygen is mandatory, lack of oxygen results in accumulation of NADH and subsequent increase in fluorescence intensity. The fluorescence intensity is, for instance, imaged using sensitive photographic or video techniques and can be used to study the regional heterogeneity of tissue dysoxia on organ surfaces in vitro and in vivo. Unwanted influence of the absorbance of hemoglobin can be corrected by use of a two-wavelength method (Coremans, 1997).
However, even with proper calibration, exact quantification of the NADH levels remains impossible (Masters, 1993). One of the reasons is the contribution of cytosolic NADH and NADPH to the total fluorescence signal.
Hence, although oxygen is one of the most important biological molecules, concentration measurements in vivo remain cumbersome. The same kinds of problems arise when the concentration of another substance is measured.